This invention relates to Dendritic Cell Transmembrane Serine Protease (DCTSP), a new member of the Type II Transmembrane Serine Protease polypeptide family, and to methods of making and using DCTSP polypeptides.
Type II Transmembrane Serine Protease (TTSP) polypeptides are related, membrane-anchored polypeptides that are involved in cell surface proteolysis and share common structural features including a proteolytic domain, a stem region comprising varying modular structural domains, a transmembrane domain, and a short cytoplasmic domain (Hooper et al., J. Biol. Chem. 276:857, 2001). Members of this family include hepsin (Leytus et al., Biochemistry 27:1067, 1988), enteropeptidase (also referred to as enterokinase; Kitamoto et al., Biochemistry 34:4562, 1995), TMPRSS2 (Paoloni-Giacobino et al., Genomics 44:309 1997), human airway trypsin-like protease (HAT; Yamaoka et al., J. Biol Chem. 273:11895, 1998), corin (Yan et al. J. Biol. Chem. 274:14296, 1999), MT-SP1 (also known as matriptase; Lin et al., J. Biol. Chem. 274:18231, 1999), and TMPRSS4 (Wallrapp et al., Cancer Res. 60:2602, 2000). Kim et al. (Biochim. Biophys. Acta. 1518:204, 2001) disclose cDNAs encoding proteins with putative serine protease domains and potential regulatory domains; one of the putative proteins also had a transmembrane domain.
The proteolytic domains of TTSPs exhibit a high degree of homology, with highly conserved motifs comprising histidine, aspartate and serine residues thought to be necessary for catalytic activity. A conserved activation motif contains an arginine or lysine, and indicates that the TTSPs are likely to be activated following cleavage. The presence of conserved cysteine residues, and their predicted disulphide bonding pattern, provide support for the belief that TTSPs are likely to remain associated with the cell membrane even after cleavage/activation, although soluble forms of some TTSPs have been identified (Hooper et al., supra). Additional conserved cysteine residues appear to be involved in forming disulphide bonds within the catalytic domain. Cleavage specificities and potential substrates have been identified for some TTSPs, but remain unknown for most; it is likely that the substrate(s) for TTSPs preferentially contain an arginine or lysine in the P1 amino acid position (as originally described for serine proteases in Schecter et al., Biochem. Biophys. Res. Commun. 27:157, 1967).
A hydrophobic, transmembrane domain is present near the N-terminus of the members of the TTSP family, indicating that the proteolytic domain is extracellular. The presence of the catalytic domain on the outside of cell, but presumably still in association with the cell membrane, suggests a role for this family of serine proteases in regulated release of substrate proteins from the cell surface, either from the same cell upon which the TTSP is found or from a cell that is in close association with such a cell.
Most TTSPs exhibit relatively restricted expression patterns, indicating that they may carry out tissue-specific functions (Hooper et al., supra). The variability of the length of the cytoplasmic domains of TTSPs renders it difficult to predict a role for these proteins in cellular signaling; however, some TTSPs do contain consensus phosphorylation sites within the cytoplasmic domain. The greatest degree of variability between members of the TTSP family occurs in the stem region, which may contain up to eleven structural domains. The variety in number and type of structural domains present in the stem region suggests that it may serve to regulate the activity and/or binding of TTSPs to substrates. The role of cell surface proteolysis in homeostasis and disease demonstrates that there is a need in the art to identify and characterize additional members of the TTSP family.
The present invention provides a novel serine protease, referred to as human Dendritic Cell Transmembrane Serine Protease (DCTSP), a Type II transmembrane protein depicted in SEQ ID NOs:1 and 2; a splice variant having 446 amino acids was also isolated; the nucleotide and amino acid sequence of this variant are shown in SEQ ID NOs:3 and 4. Also provided are DCTSP polypeptides that are encoded by nucleic acids capable of hybridizing to the DNAs of SEQ ID NOs:1 or 3 and that have at least one DCTSP activity, DCTSP polypeptides that are at least 80% identical to the DCTSP polypeptides of SEQ ID NOs: 2 or 4 and that have at least one DCTSP activity, and fragments of such DCTSP polypeptides that have at least one DCTSP activity.
The invention further provides various forms of DCTSP, including: a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 207 to 212, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 221 to 226, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 75 to 95, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 174 to 179, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 174 to 179, inclusive, and y represents an integer from 190 to 195, inclusive; and a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 190 to 195, inclusive. Also comprehended herein are nucleic acids encoding the aforementioned DCTSP polypeptides, vectors comprising such nucleic acids, host cells transformed or transfected with such vectors (including hosts cells wherein the DNA encoding a DCTSP polypeptide is integrated into host cell chromosomal DNA), and processes for obtaining DCTSP polypeptides by culturing such host cells.
Soluble forms of DCTSP (including fragments comprising the extracellular domain as well as fragments comprising the cytoplasmic domain) will be useful in vitro to screen for agonists or antagonists of DCTSP activity utilizing one or more screening methods, which methods also form an aspect of the present invention. In one aspect, the inventive methods utilize homogeneous assay formats such as fluorescence resonance energy transfer, fluorescence polarization, time-resolved fluorescence resonance energy transfer, scintillation proximity assays, reporter gene assays, fluorescence quenched enzyme substrate, chromogenic enzyme substrate and electrochemiluminescence. In another aspect, the inventive methods utilize heterogeneous assay formats such as enzyme-linked immunosorbant assays (ELISA) or radioimmunoassays. In yet another aspect of the invention are cell-based assays, for example those utilizing reporter genes, as well as functional assays that analyze the effect of an antagonist or agonist on biological function(s).
The invention further provides methods for producing information comprising the identity of a compound that alters one or more activities of DCTSP, comprising using one or more of the inventive assays to identify a compound or compounds that alter the binding and/or cleavage of substrate by DCTSP. In one embodiment, the compound increases (or agonizes) the binding and/or cleavage of substrate, and in another distinct embodiment, the compound decreases (or antagonizes) the binding and/or cleavage of substrate.
Also provided by the invention is the information produced according to the inventive methods, said information comprising the identity of a compound that alters the activity of DCTSP, and preferably embodied in a storage medium selected from the group consisting of paper, magnetic tape, optical tape, floppy disks, compact disks, computer system hard drives, and computer memory units. In a further aspect, the invention provides a database comprising said information, wherein the information is preferably embodied in a computer-readable medium, and a separate embodiment wherein the information is embodied in a human-readable medium.
Additionally provided by the invention is a computer system comprising a database containing records pertaining to a plurality of compounds, wherein the records comprise results of an assay of the invention, and a user interface allowing a user to access information regarding the plurality of compounds. In another aspect of the invention, a computer system is provided for storing and retrieving data on a plurality of compounds, the computer system comprising:
input means for entering data for the compounds into a storage medium;
a processor for creating an individual record for each compound, the processor assigning specific identifying values for each compound;
means for selecting one or more of the records based on results in an assay; and
means for transmitting information in the record or records to an output device to produce a report; preferably a report in human-readable form, and wherein the computer system preferably further comprises a video display unit.
The invention also provides a method of using the computer system of the invention to select one or more compounds for testing from a plurality of compounds having records stored in a database, the method comprising: displaying a list of said records or a field for entering information identifying one or more of said records; and selecting one or more of the records from the list or the record or records identified by entering information in the field.
Further, the invention provides a method of operating a computer system for analyzing compounds that modulate the activity of DCTSP, the method comprising:
entering data relating to a plurality of compounds into a storage medium;
processing the data to create an individual record for each compound;
testing compounds for the ability to modulate activity of DCTSP; and
communicating results from the testing into the storage medium such that results for each compound are associated with the individual record for that compound; wherein in one embodiment the storage medium comprises one or more computer memory units, and in another embodiment the computer system further comprises a video display unit.
In yet another aspect of the invention, a database is provided comprising records generated according to the methods of the invention, and a method is provided for selecting compounds that modulate the activity of DCTSP, comprising compiling said database, analyzing the testing results, and selecting one or more compounds.